Targeted donor dna insertion and indel editing of plant genes

Abstract

The disclosure relates to methods of producing genome edited plant cells, genome edited plant calli, and genome edited plants with genome edits at multiple genomic sites. The disclosure also relates to systems and kits for producing genome edited plant cells with genome edits at multiple genomic sites. Disclosed methods include those where a guide RNA (gRNA) directed to a first genomic DNA site, a donor DNA template polynucleotide, and a polynucleotide comprising a DNA molecule encoding a second gRNA directed to a second genomic DNA site are introduced into a plant cell and genome edited cell having an insertion of the donor DNA template polynucleotide or fragment thereof in the first genomic DNA site and a DNA modification at the second genomic DNA site is selected.

What is claimed is: 
     
         1 . A method of producing a genome edited plant cell comprising:
 (a) introducing into a plant cell a first guide RNA (gRNA) directed to a first genomic DNA site, a donor DNA template polynucleotide, and a polynucleotide comprising a DNA molecule encoding a second gRNA directed to a second genomic DNA site; wherein the plant cell comprises at least one Cas nuclease and/or one or more polynucleotides encoding the Cas nuclease(s); and   (b) selecting for the genome edited plant cell, wherein the genome edited plant cell comprises a first insertion of the donor DNA template polynucleotide or fragment thereof in the first genomic DNA site and a DNA modification at the second genomic DNA site comprising a second insertion, a deletion, and/or a substitution of one or more nucleotides, and wherein the second insertion does not comprise the donor DNA template polynucleotide or fragment thereof.   
     
     
         2 . The method of  claim 1 , further comprising the step of regenerating a genome edited plant callus and/or a genome edited plant from the genome edited plant cell of step (b). 
     
     
         3 . The method of  claim 1 , wherein the plant cell comprising the Cas nuclease comprises a polynucleotide encoding the Cas nuclease. 
     
     
         4 . The method of  claim 1 , wherein the polynucleotide encoding the Cas nuclease expresses the first Cas nuclease in the plant cell, optionally wherein the polynucleotide encoding the Cas nuclease expresses the first Cas nuclease in the plant cell before introducing the first gRNA, donor DNA template polynucleotide, and the polynucleotide of in step (a). 
     
     
         5 . The method of  claim 1 , wherein the Cas nuclease is provided as a ribonucleoprotein (RNP) complexed with the first gRNA and as a polynucleotide comprising a DNA molecule that encodes the Cas nuclease. 
     
     
         6 . The method of  claim 5 , wherein the polynucleotide comprising the DNA molecule encoding the second gRNA further comprises a DNA molecule encoding a Cas nuclease which recognizes the second gRNA. 
     
     
         7 . The method of  claim 1 , wherein the plant cell comprises at least two distinct Cas nucleases, optionally wherein the two distinct Cas nucleases comprise a first Cas nuclease which recognizes the first gRNA and a second Cas nuclease which recognizes the second gRNA. 
     
     
         8 . The method of  claim 1 , wherein the Cas nucleases or one or more polynucleotides encoding the Cas nuclease(s) are introduced before and/or with the first gRNA, donor DNA template polynucleotide, and the polynucleotide comprising a DNA molecule. 
     
     
         9 . The method of  claim 1 , wherein at least two distinct Cas nucleases and/or one or more polynucleotides encoding at least one of the Cas nucleases are introduced into the plant cell in step (a), optionally wherein a first Cas nuclease which recognizes the first gRNA and a second Cas nuclease recognizes the second gRNA. 
     
     
         10 . The method of  claim 9 , wherein the two distinct Cas nucleases comprise a first and a second Cas nuclease, wherein the first Cas nuclease recognizes the first gRNA and is introduced as an RNP and wherein a polynucleotide encoding the second gRNA and a second Cas nuclease which recognizes the second gRNA is introduced. 
     
     
         11 . The method of  claim 1 , wherein the Cas nuclease(s) comprise a Type II and/or a Type V Cas nuclease. 
     
     
         12 . The method of  claim 1 , wherein the introducing of step (a) is by bacterially-mediated transformation, optionally wherein the bacterially-mediated transformation is  Agrobacterium -mediated transformation. 
     
     
         13 . The method of  claim 1 , wherein the introducing of step (a) is by biolistics, electroporation, and/or transfection. 
     
     
         14 . The method of  claim 1 , wherein the introducing of step (a) is into callus comprising the plant cell. 
     
     
         15 . The method of  claim 1 , wherein the introducing of step (a) is into an explant comprising the plant cell. 
     
     
         16 . The method of  claim 1 , wherein the donor DNA template polynucleotide lacks homology arms complementary to DNA at the first genomic site. 
     
     
         17 . The method of  claim 16 , wherein the donor DNA template polynucleotide is integrated by non-homologous end-joining (NHEJ). 
     
     
         18 . The method of  claim 1 , wherein the donor DNA template polynucleotide comprises homology arms complementary to DNA at the first genomic site. 
     
     
         19 . The method of  claim 18 , wherein a fragment of the donor DNA template polynucleotide is integrated at the first genomic DNA site by homology-directed repair (HDR). 
     
     
         20 . The method of any one of  claims 1 to 19 , wherein the plant cell comprises one or more homology-dependent repair (HDR)-enhancing polypeptides, optionally wherein the HDR-enhancing polypeptides comprise a bacteriophage lambda exonuclease, a bacteriophage lambda beta single-stranded annealing protein (SSAP), and/or an  E. coli  single stranded binding protein (SSB). 
     
     
         21 . The method of any one of  claims 1 to 19 , wherein the selecting comprises assaying plant cells obtained from step (a) for the first insertion of the donor DNA template polynucleotide or fragment thereof in the first genomic DNA site and/or for the DNA modification at the second genomic DNA site comprising a second insertion, a deletion, and/or a substitution of one or more nucleotides to identify a plant cell comprising the first insertion and/or the DNA modification. 
     
     
         22 . The method of  claim 21 , wherein the assaying comprises a phenotypic assay, a nucleic acid amplification-based assay, a nucleic acid hybridization-based assay, and/or a sequencing-based assay for the insertion and/or the DNA modification. 
     
     
         23 . The method of  claim 21 , wherein the identified plant cell is propagated or regenerated to form callus and or a plant comprising the first insertion and/or the DNA modification. 
     
     
         24 . The method of any one of  claims 1 to 19 , wherein the plant cell is a maize plant cell, a wheat plant cell, or a soybean plant cell. 
     
     
         25 . A plant cell genome editing system comprising a target plant cell containing a first guide RNA (gRNA) directed to a first genomic DNA site, a donor DNA template polynucleotide, a polynucleotide comprising a DNA molecule encoding a second gRNA directed to a second genomic DNA site, and at least one Cas nuclease and/or one or more polynucleotides encoding the Cas nuclease(s), wherein the system provides a genome edited plant cell comprising a first insertion of the donor DNA template polynucleotide or fragment thereof in the first genomic DNA site and a DNA modification at the second genomic DNA site comprising a second insertion, a deletion, and/or a substitution of one or more nucleotides, wherein the second insertion does not comprise the donor DNA template polynucleotide or fragment thereof. 
     
     
         26 . The system of  claim 25 , wherein the system provides for recovery of the genome edited plant cell from the target plant cell in a single selection. 
     
     
         27 . The system of  claim 25 , wherein the genome edited plant cell is obtained from the target plant cell without first selecting for a plant cell comprising only the insertion at the first genomic site or only the modification at the second genomic site. 
     
     
         28 . The system of  claim 25 , wherein the Cas nuclease is provided as a ribonucleoprotein (RNP) complexed with the first gRNA and as a polynucleotide comprising a DNA molecule that encodes the Cas nuclease. 
     
     
         29 . The system of  claim 25 , wherein the plant cell comprises at least two distinct Cas nucleases, optionally wherein the two distinct Cas nucleases comprise a first Cas nuclease which recognizes the first gRNA and a second Cas nuclease which recognizes the second gRNA. 
     
     
         30 . The system of  claim 29 , wherein at least two distinct Cas nucleases and/or one or more polynucleotides encoding at least one of the Cas nucleases are introduced into the plant cell in step (a), optionally wherein a first Cas nuclease which recognizes the first gRNA and a second Cas nuclease recognizes the second gRNA. 
     
     
         31 . The system of  claim 29 , wherein the two distinct Cas nucleases comprise a first and a second Cas nuclease, wherein the first Cas nuclease recognizes the first gRNA and is introduced as an RNP and wherein a polynucleotide encoding the second gRNA and a second Cas nuclease which recognizes the second gRNA is introduced. 
     
     
         32 . The system of  claim 25 , wherein the Cas nuclease(s) comprise a Type II and/or a Type V Cas nuclease. 
     
     
         33 . The system of  claim 25 , wherein the donor DNA template polynucleotide lacks homology arms complementary to DNA at the first genomic site. 
     
     
         34 . The system of  claim 33 , wherein the donor DNA template polynucleotide is integrated at the first genomic DNA site by non-homologous end-joining (NHEJ). 
     
     
         35 . The system of  claim 25 , wherein the donor DNA template polynucleotide comprises homology arms complementary to DNA at the first genomic site. 
     
     
         36 . The system of  claim 35 , wherein a fragment of the donor DNA template polynucleotide is integrated at the first genomic DNA site by homology-directed repair (HDR). 
     
     
         37 . The system of  claim 25 , wherein the target plant cell further comprises one or more homology-dependent repair (HDR)-enhancing polypeptides, optionally wherein the HDR-enhancing polypeptides comprise a bacteriophage lambda exonuclease, a bacteriophage lambda beta single-stranded annealing protein (SSAP), and/or an  E. coli  single stranded binding protein (SSB). 
     
     
         38 . The system of  claim 25 , further comprising an assay to identify the genome edited plant cell. 
     
     
         39 . The system of  claim 25 , wherein the genome edited plant cell is selected from a population of the target plant cells with the assay. 
     
     
         40 . The system of  claim 39 , wherein the assay comprises a phenotypic assay, a nucleic acid amplification-based assay, a nucleic acid hybridization-based assay, and/or a sequencing-based assay for the insertion and/or the DNA modification. 
     
     
         41 . The system of  claim 39 , wherein the system further comprises a plant tissue culture system for regenerating callus or a plant from the genome edited plant cell. 
     
     
         42 . The system of  claim 25 , wherein the plant cell is a maize plant cell, a wheat plant cell, or a soybean plant cell. 
     
     
         43 . A kit comprising:
 (a) a first guide RNA (gRNA) directed to a first genomic DNA site in a plant cell and a donor DNA template polynucleotide; and,   (b) a polynucleotide comprising a DNA molecule encoding a second gRNA directed to a second genomic DNA site in a plant cell.   
     
     
         44 . The kit of  claim 43 , wherein the kit further comprises at least one Cas nuclease that recognizes at least one of the gRNAs. 
     
     
         45 . The kit of  claim 44 , wherein the first gRNA is complexed with a Cas nuclease as a ribonuclear protein (RNP). 
     
     
         46 . The kit of  claim 43 or 44 , wherein the DNA molecule encodes a Cas nuclease. 
     
     
         47 . The kit of  claim 46 , wherein the Cas nuclease in the RNP is distinct from the Cas nuclease encoded by the DNA molecule. 
     
     
         48 . The kit of  claim 44 , wherein the Cas nuclease(s) comprise a Type II and/or a Type V Cas nuclease. 
     
     
         49 . The kit of  claim 43 , wherein the donor DNA template polynucleotide lacks homology arms complementary to DNA at the first genomic site. 
     
     
         50 . The kit of  claim 43 , wherein the donor DNA template polynucleotide comprises homology arms complementary to DNA at the first genomic site. 
     
     
         51 . The kit of  claim 43 , wherein the kit further comprises a plant cell containing the first and the second genomic sites, optionally wherein the plant cell is a maize plant cell, a wheat plant cell, or a soybean plant cell.